1.

Introduction

Smoking tobacco cigarettes has been a known health hazard since the late 1950s.¹ Smoking is a risk factor for numerous respiratory and cardiovascular conditions that lead to serious diseases and mortality. Two main respiratory health concerns arising from smoking are lung cancer, due to the inhalation of toxins and exposure of carcinogenic by-products of combustion, and chronic obstructive pulmonary disease (COPD), which is associated pathologically with inflammation, gas trapping, and emphysema, along with remodeling of the small airways and pulmonary vasculature.

To investigate the disease pathogenesis of COPD in vivo requires modeling in rodents. Models have emerged using guinea pigs^2,3 and mice^4–11 with COPD being induced by exposure to the chemicals found in cigarette smoke over a period of up to six months. In guinea pigs, histological analysis has demonstrated that airway remodeling and emphysema can be stopped or slowed by treatment with a myeloperoxidase inhibitor,² which also prevents the muscularization of small intrapulmonary arteries. Wright et al. reported the use of a guinea pig model to investigate smoke-induced pulmonary hypertension, endothelial dysfunction, emphysema and small airway remodeling.³ Mouse models have shown pulmonary inflammation as measured by histology and in bronchoalveolar lavage.^7,8

To investigate the lungs in 3D, high-resolution micro computed tomography (micro-CT) can be used on fixed specimens, and in vivo micro-CT machines can image live rodents. To investigate the lungs at a specified respiratory phase, prospectively gated imaging allows for each projection view to be acquired at the same point in the respiratory cycle,^12–16 whereas retrospective approaches select only the desired in-phase projections for reconstruction.^17–22 Parameswaran et al.⁵ fixed sections of mouse lung tissue and stained with silver to provide contrast in the micro-CT images in order to measure the alveolar airspace volume. Using an in vivo micro-CT scanner, the lung volume, CT density, and low attenuation regions can be quantified in live animals.^4,9,11 Recent papers have reported using respiratory-gated¹⁰ or respiratory- and cardiac-gated micro-CT imaging⁶ during the end expiratory phase to improve the measurements of air trapping. Other researchers have used in vivo micro-CT imaging to study emphysema in a knock-out model²³ or in an elastase-exposure model.^24,25

The majority of recent studies discussed here focus on either the small airway remodeling in excised tissue or on the low attenuation regions and gas trapping in vivo as markers of emphysema. To further understand COPD and how this disease progresses, longitudinal in vivo studies of the airways, lung function, and pulmonary vasculature together using noninvasive 3D imaging will become necessary. In this paper, we perform longitudinal in vivo micro-CT in mice that had been exposed to cigarette smoke for different durations up to six months. The micro-CT images will provide anatomical and functional measurements of the lungs and airways and will be correlated with histological assessment of the excised lung tissues.

2.

Methods

3.

Results

Images of a representative mouse from each time point of the smoke-exposed group are shown in Fig. 1, along with a baseline image. These images were obtained during end-expiration. Note the air-filled regions in the lungs appear darker following 6 months of smoke-exposure [Figs. 1(c) and 1(g)] compared with the shorter exposure durations. The mean CT numbers for these mice were $-288\mathrm{HU}$ for the 1-month exposure [Figs. 1(a) and 1(e)], $-253\mathrm{HU}$ for the 3-month exposure [Figs. 1(b) and 1(f)], −351 HU for the 6-month exposure [Figs. 1(c) and 1(g)], and $-267\mathrm{HU}$ for the baseline images [Figs. 1(d) and 1(h)]. Figure 2 shows segmentations of the lung (below 0 HU), the mean attenuation (below $-600\mathrm{HU}$), and the low attenuation (below $-900\mathrm{HU}$) in the inspiration phase. The top row shows the smoke-exposed mouse and the bottom row shows an age-matched air-exposed mouse looking from the sternum and from the right side of the animal. The uneven distribution of gray scale values shows the patchy regional inhomogeneities in the lung ventilation that is typical in COPD.

Fig. 1

Axial (top) and coronal (bottom) slices from images taken following (a), (e) 1 month; (b), (f) 3 months; and (c), (g) 6 months of smoke-exposure. (d), (h) Baseline images for the same mouse as the 6-month exposure. Images were obtained during end-expiration and reconstructed with 0.075-mm voxel spacing.

Fig. 2

Segmentations of the lung in white, average attenuation regions (below $-600\mathrm{HU}$) in green and low attenuation regions (below $-900\mathrm{HU}$) in dark green from inspiration images taken following (a), (b) 6 months of smoke-exposure and (c), (d) 6 months of air-exposure. The air-exposed mouse has a more uniform distribution of the low attenuation regions compared with the smoke-exposed mouse.

Measurements of the lung and major airway volumes, and the corresponding lung and major airway densities were made in the baseline and endpoint images for both respiratory phases, whereas the %LAA was measured in the inspiration images only (Table 1). The measurements from the two respiratory phases were combined to calculate the functional metrics: Vt, FRC, and Vt/RI ratio (Table 2). Statistically significant differences between baseline and endpoint are shown in bold font for both tables. During inspiration, both the smoking and control mice exhibited significant increases in lung volume for the 6-month ($p<0.0001$ for both) and 3-month ($p=0.0033$ for smoking and $p<0.0001$ for control) exposure groups. The 1-month smoked-exposed mice also showed increased lung volume ($p=0.0003$), whereas the control group showed an increase in major airway volume ($p=0.0054$) and %LAA ($p=0.0007$), which may be due to the small volumes measured at baseline. The 6-month exposure groups were the only groups to show a significant increase in FRC ($p<0.0001$ for smoking and $p=0.0296$ for control). The 6-month smoking group was the only group with no change in Vt and a decrease in Vt/RI.

Table 1

Volumetric measurements and CT densities measured before and after the exposures. Mean and standard deviations for the lung volume, lung density, major airway volume, and % LAAs.

Table 2

Functional metrics measured before and after the exposures. Mean and standard deviations for body weight, FRC, Vt, and the ratio of Vt to RI volume (Vt/RI).

To account for the biological variability and the growth of the mice during the study, the metrics we compared were the changes over the course of the exposure period (endpoint to baseline), where positive changes indicate an increase over the exposure period and negative values indicate a reduction in the measurements over the exposure period. The changes in the measured values are given in Table 3, with bold font indicating statistically significant differences between the smoke-exposed and air-exposed mice for the same exposure duration. The 6-month smoke-exposure group showed a significantly larger increase in lung volume at end expiration ($p=0.0025$) than age-matched controls. The 3-month smoke-exposed mice showed an increase in the air content in the lungs during inspiration ($p=0.0198$) and an increase in the major airway volume during end expiration ($p=0.0251$). The changes in the calculated functional metrics (endpoint to baseline), along with body weight, are plotted as Fig. 3, with the $p$-values included on the plot. The histograms for inspiration and expiration are shown in Fig. 4, with the baseline values shown as squares, and the endpoint values as circles. Each data point is the mean value for the exposure group with the standard error in the mean shown as errorbars. From these curves, there is a lot of overlap between the groups. The 6-month smoke-exposed group (green) shows a deviation to higher counts and lower CT #, indicating that there is increased air in the lung at both respiratory phases. This increased air is consistent with air-trapping, a clinical sign of COPD.

Table 3

Change in lung measurements from baseline to endpoint: mean and standard deviations for changes in lung and major airway volumes, and the corresponding CT density taken during end expiration (top) and inspiration (bottom). The % LAA is also given during inspiration. Values are the endpoint measurements minus baseline for the same mouse, averaged for the group. Bold entries indicate statistically significant differences between the smoke-exposed and age-matched air-exposed mice.

Fig. 3

Mean differences between the baseline and endpoint scans showing the changes in (a) body weight, (b) ratio of Vt/RI, (c) FRC, and (d) Vt. P-values are shown for comparisons between smoke-exposed and air-exposed mice. Vt, tidal volume; RI, resting inspiration; and FRC, functional residual capacity.

Fig. 4

Histograms of the CT number in Hounsfield units within the lungs. The curves show the mean and standard error for the baseline images (squares) and the endpoint images (circles). The smoke-exposed mice are shown during (a) inspiration and (b) expiration, and the air-exposed mice during (c) inspiration and (d) expiration.

Measurements on the histological slides were completed for air-exposed mice with $n=5$ for 1-month, $n=7$ for 3-month, and $n=6$ for 6-month exposure, and for smoke-exposed mice with $n=7$ for 1-month and 3-month and $n=11$ for 6-month exposures. Sample images from each exposure group are shown in Fig. 5 for H&E staining, which demonstrates airway disruptions for the smoke-exposed mice starting at 3 months. Airway disruptions are a degradation of the lung tissue, which indicates emphysema and air trapping in the lungs. Although the volumetric measurements done in the CT images are not sensitive enough to pick up the early stage changes in the lung, the 6-month smoke exposure group shows air-trapping, which is consistent with the histological results. The mean linear intercept is shown in Fig. 5(d), with a statistically significant difference observed between the air-exposed and smoke-exposed mice following the 6-month exposure time. Figure 6 shows collagen in a representative airway stained with Masson’s Trichrome. Plots are shown in Fig. 6(d) for the total collagen intensity, with no significant differences found between the groups. Measurements were performed in the peripheral airways, with airway diameters down to $200\mu \mathrm{m}$. The histological results are consistent with other studies,^2,26,31 which confirms that the smoke-exposures were successful in inducing the COPD model as desired.

Fig. 5

H&E stained slides for air-exposed and smoke-exposed mice following exposures of (a) 1 month, (b) 3 months, (c) 6 months. (d) Plot of mean linear intercept. $P$-values are shown for comparisons between smoke-exposed and air-exposed mice.

Fig. 6

Trichrome stained slides for air-exposed and smoke-exposed mice following exposures of (a) 1 month, (b) 3 months, (c) 6 months. (d) Plot of total collagen intensity. $P$-values are shown for comparisons between smoke-exposed and air-exposed mice.

Outlier assessment using the Grubbs identified a few outliers (no more than 1 per exposure group) for a few of the measurements. These outliers were mainly a result of artifacts in the image. For a couple of scans, we noted some dark ring artifacts that were unfortunately located over the lung that we were unable to exclude from the analysis. In other images, we had a few streak artifacts caused by changes in the respiratory pattern during the scan. Eliminating these as outliers improved the confidence in the measured values as representing the organ or tissue of interest.

4.

Discussion

The mice were quite young at the beginning of the exposure, and therefore still growing, especially over the first couple of months. The young age at initiation of exposure mimics the human use of cigarettes, where smoking often begins in adolescence. As a result of the mice continuing to develop during the early stage of the exposures, the measured values obtained from the micro-CT imaging data were somewhat skewed when compared to the baseline scans, as increased measured values were due to both age-related growth and to the exposure received. To account for the age-related growth, we only compared age-matched animals and to account for biological variation in animal size, we measured the change in each metric relative to baseline for individual mice to promote consistency in the data.

We found significant reductions in weight gain for the smoke-exposed mice compared with air-exposed mice following 3 or 6 months of exposure ($p<0.0001$). All mice gained weight, but the smoke-exposed mice gained less weight than their air-exposed counterparts. As the duration of smoke-exposures increased, the weight gain was reduced.

The change in the lung volume measurements during end-expiration was increased in the 6-month smoke-exposed mice compared to air-exposed mice ($p=0.0025$). This pattern was also seen in the FRC measurements, with smoke-exposed mice exhibiting a larger increase in FRC than can be expected by age-related growth alone ($p=0.0049$). The change in the Vt/RI ratio in the 6-month smoke-exposed mice was significantly reduced compared to the air-exposed mice ($p=0.0044$). The histogram analysis of the air content in the lungs also showed an increase in air content for the 6-month smoke-exposed animals for both respiratory phases and a noticeable shift in the peak of the curve during expiration. Together, these metrics (lung volume during end-expiration, FRC, Vt/RI, and histogram) suggest air-trapping in the lung likely related to emphysema and/or airway remodeling. Furthermore, the increase in Lm in the mice exposed for 6 months to cigarette smoke is consistent with measurements reported by Zhou et al.³² Histological images of the 6-month time point show a similar pattern of centrilobular emphysema in published studies.³¹

The image-based measurements obtained from the micro-CT scans are whole-organ measurements, similar to the types of analyses previously published.^6,23,25 One novel aspect of this study is the inclusion of both end-expiration and peak inspiration images and the use of free-breathing animals, which allows for estimates of lung function. The functional micro-CT measurements given here are similar to what is performed in the clinical evaluation of COPD. However, the inspiratory to total lung capacity (IC/TLC) metric that is used clinically is underestimated in anaesthetized and free-breathing mice, as we can only measure the average inhalation during tidal breathing, not the maximum inhalation, for the IC measurement. As a surrogate, we tabulated the Vt to resting inspiration ratio (Vt/RI) instead and showed significant differences in the exposure groups at 6 months.

Limitations to this study include the ability to compare directly with functional metrics obtained using other methods, such as pulmonary function testing with a ventilator. Using a ventilator would enable the researcher to fill the lungs to the inspiratory capacity and make accurate measurements of the total lung capacity. Forcing the lungs to the total lung capacity provides more information about tissue elasticity, respiratory resistance, etc., that are not possible using free-breathing mice. Furthermore, metrics that are used clinically, such as the IC/TLC can be estimated, providing a more direct linkage between preclinical studies and patient outcomes. However, this maneuver requires intubation and mechanical ventilation of the mouse, which can lead to damage and inflammation in the trachea or overinflation of the lungs³³ that will result in measured volumes that are over-estimated in the micro-CT images.

The 1-month and 3-month exposure groups did not show significant differences for metrics related to COPD. The development of COPD is not uniform throughout the lung parenchyma. Since the micro-CT metrics FRC, Vt, %LAA, and Vt/RI are based on whole-lung measurements, small regional changes may be lost. In reality, the smoke-exposures may not induce measurable effects until later time points, which have been suggested in the literature^8,32 for histological measurements. Some COPD metrics were identified for the 6-month exposure group, which suggests that in vivo micro-CT imaging is sensitive to these markers.

This study represents the first in vivo functional micro-CT imaging study of smoke-exposed mice with different exposure durations. Although other researchers have published micro-CT studies of smoke-induced or elastase-induced emphysema, they focused on a single phase in the respiratory cycle,^6,10 or were ungated,^4,9,11 or used breath-hold during the scans.^24,25 Images taken from a single respiratory phase can only assess lung volume and air content at that phase, so functional information is lost, whereas ungated images provide an average lung size and air content over the full cycle. Breath-hold imaging can be done at multiple phases, but the lungs of rodents are often over-inflated during mechanical ventilation,³³ so any functional information will suffer from a systematic error. By performing the micro-CT imaging at multiple phases in the cycle in free-breathing mice, as described in this study, we can add important functional metrics, such as Vt and Vt/RI ratio, to the analysis.

In this study, we demonstrate that in vivo respiratory-gated micro-CT obtained at multiple respiratory phases can provide information about the lung structure and function of free-breathing mice in a model of COPD. This noninvasive technique will allow preclinical researchers to use imaging techniques consistent with the clinical diagnosis of COPD, image mice at multiple time points within a study, and will enable further research to assess new therapeutic treatments in vivo.